www.impactjournals.com/oncotarget/ Oncotarget, 2017, Vol. 8, (No. 12), pp: 19934-19946 Research Paper CBX3 promotes colon cancer cell proliferation by CDK6 kinase- independent function during cell cycle Yao Fan1,2, Haiping Li1,2, Xiaolong Liang1,2, Zheng Xiang1,2 1Chongqing Key Laboratory of Department of General Surgery, Chongqing Medical University, Chongqing, China 2Department of Gastrointestinal Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China Correspondence to: Zheng Xiang, email: [email protected] Keywords: colon cancer, CBX3, CDK6, p21, proliferation Received: August 01, 2016 Accepted: December 12, 2016 Published: February 10, 2017 ABSTRACT Heterochromatin protein 1γ (CBX3) links histone methylation marks to transcriptional silence, DNA repair and RNA splicing, but a role for CBX3 in cancer remains largely unknown. In this study, we show that CBX3 in colon cancer cells promotes the progression of the cell cycle and proliferation in vitro and in vivo. Cell cycle (G1 phase to S phase) related gene CDK6 and p21 were further identified as targets of CBX3. In addition, we found that enhancing CDK6 suppresses cell proliferation by upregulating inhibitor p21 in the absence of CBX3, and this function is independent of the kinase activity of CDK6. Our results demonstrate a key role of CBX3 in colon carcinogenesis via suppressing the expression of CDK6/p21, which may disrupt the role of CDK6 in transcriptionally regulating p21, as part of a negative feedback loop to limit CDK6 excessive activation. INTRODUCTION Cyclin dependent kinases (CDKs) are core parts of cell cycle regulation, containing over 20 members [16]. The histone proteins as well as several other proteins CDKs in binding and catalyzing cyclins are necessary to are essential elements of heterochromatin formation, and drive progression of the cell cycle. CDK4/CDK6-cyclinD are associated with gene transcriptional repression or complexes enable the cells to pass from G1 to S phase, silencing [1]. The mammalian heterochromatin protein 1 but these proteins are critically required for proliferation is a highly conserved non-histone chromosomal protein only in selected cells types [17, 18]. Experiments with and a major component of heterochromatin. Its orthologs gene-deficient mice for CDK4 or CDK6 are viable [19], include CBX5, CBX3 and CBX1, which are concentrated then lacking CDK4 and CDK6 die of severe anemia and at pericentric heterochromatin or euchromatic sites, hematopoietic abnormalities [20]. Partial experiments each containing a chromodomain [2]. Methylation of demonstrate D-cyclins enhancing interaction with CDK2 histone H3 lysine 9 (H3K9) can be recognized by the or cyclinD-CDK4/CDK6-independent mechanism. CBX3 chromodomain [3]. Dynamic CBX3 binding to However, several are also involved in transcriptional H3K9 is dependent on the histone methyltransferase regulation, DNA damage repair, stem-cell self-renewal, Suv339H1 and maintains stable heterochromatin domains metabolism, spermatogenesis and neuronal function to regulate gene expression [4]. In addition, CBX3 can [21]. Recent researches focus on the role of CDK6 in recruit various cofactors which perform functions in transcriptional regulation. CDK6 acts as a cofactor intracellular biological processes by binding methylated targeting gene promoter, including IL-1, IL-8, EGRI [22– H3K9. These functions include RNA alternative splicing, 25]. The most prominent report that a kinase-independent DNA damage response, transcription elongation, cell function of CDK6 links cell cycle to tumor angiogenesis growth and differentiation [5–10]. Recently, although gene shows CDK6 participating in p16 and VEGF-A expression assays have revealed that CBX3 is linked with transcriptional regulation in lymphoid malignancies [26], lung cancer, osteosarcoma, gastric cardia adenocarcinoma CDK6 acts as a transcriptional regulator on the p16INK4a and colorectal cancer, among other diseases [11–15], the promoter providing an internal safeguard by negative specific mechanisms by which CBX3 promotes cancer feedback loop. However, CDK6 kinase-independent progression are poorly understood. function in colorectal cancer has never been reported. www.impactjournals.com/oncotarget 19934 Oncotarget To investigate the mechanism by which CBX3 plasmid was constructed as shown in (Supplementary promotes colon cancer, we deleted CBX3 in HCT116 with Figure 1). Several colony cell lines were established by the CRISPR/Cas9 editing system and found that cell cycle from transfected cells. We found that the expression of progression was curbed in G1 phase and proliferation was CBX3 in the cell line, HCT116-B3 was significantly inhibited. Gene expression analysis reveals that CDK6/ lower than that in others (Figure 1A, 1B, 1C and 1D). p21 is upregulated. In addition, CDK6 is able to directly Furthermore, no fluorescence was detected in HCT116-B3 regulate p21 expression in a kinase-independent manner. cells by cell immunofluorescence when immunostained Therefore, we identify a new mechanism by which CBX3 with anti-CBX3 fluorescence antibody (Figure 1E). To promotes colon cancer progression via the CDK6/p21 verify that the deletion of CBX3 was due to CRISPR/cas9 pathway during cell cycle. editing, CBX3 genome was sequenced in HCT116-WT, B1, B3 and B4 cells. We found that an additional base T RESULTS was inserted in the NGG site to change the CBX3 genome sequence (Figure 1F). CRISPR/Cas9 induced CBX3 genome mutation CBX3 promoted colon cancer cell cycle To identify the relationship between CBX3 and the progression and proliferation in vitro pathological process of colon cancer, the CRISPR/cas9 genome editing system was used to modulate the CBX3 To investigate the effect of the deletion of CBX3 gene in HCT116 cells. sgRNA was designed and Px330 on colon cancer cell growth, we used flow cytometry to Figure 1: CRISPR/cas9 editing system induced the deletion of CBX3 protein. A. Quantitative real-time PCR (qRT-PCR) analysed mRNA level of CBX3 in HCT116-B1, B2, B3, B4 and B5 colony cell lines. The mean±SD were shown from three independent experiment, **P<0.01 compared with control. B. Immunoblot for CBX3 of five colony cell lines derived from transfecting px330 plasmid in which Lmnb1 served as a loading control. C. qRT-PCR analysis mRNA level of CBX3 from HCT116-B3 cell lines continuously cultured for 5 generations. The mean±SD were shown from three independent experiments. **P<0.01 compared with the control. D. Immunoblot for CBX3 of HCT116-B3 and HCT116-WT cells, ACTB served as a loading control. E. Cell immunofluorescence was performed in HCT116-B3 and HCT116-WT cells. Nuclei were identified by DAPI, immunofluorescence staining for CBX3. Scale bar=10 um.F. Base T was inserted in NGG site in B1, B3 and B4, base G mutation is base A in B4, verified by sequencing the CBX3 genome. www.impactjournals.com/oncotarget 19935 Oncotarget analyze cell cycle and apoptosis. These results shown after incubation for 5 days (Figure 2C and Supplementary that cell cycle was significantly inhibited in G1 phase Figure 3B). These results were further confirmed by compared with the control (Figure 2A and Supplementary EdU staining which could detect nucleotide analogue Figure 3A), but we did not find any change in apoptosis incorporation into replicated DNA (Figure 2D). These (Supplementary Figure 2A and 2B). Cell proliferation was data indicate that CBX3 promotes colon cancer cell also analyzed by MTS assay, and the deletion of CBX3 proliferation by curbing cell cycle G1-S phase transition. in colon cancer cells evidently reduced cell growth rate (Figure 2B and Supplementary Figure 3C). To directly The deletion of CBX3 directly enforces the observe cell growth, colony formation assays were expression of CDK6 and p21 performed. A specified number of cells were seeded into plates and the deletion of CBX3 in the colon cancer cell To investigate the underlying mechanism, we line significantly reduced the numbers of colonies per dish examined the expression of several genes known to be Figure 2: CBX3 deletion inhibited cell cycle progression and proliferation in vitro. A. Flow cytometry assays were performed to analyze cell cycle in HCT116-WT and B3 cells. Values at different stages of cell cycle represent mean±SD from three independent experiments. **P<0.01 compared with control. B. Colon cancer cell proliferation were detected by MTS and absorbance at 490 nm at different time points is shown. Each data point represents mean±SD from three independent experiments. **P<0.01 compared with control. C. Colony formation assays were performed using HCT116-WT and HCT116-B3 cells. Colony number were analyzed by measuring absorbance at 570 nm, The results were shown as mean±SD from three independent experiments. *P<0.05 compared with control. D. The effect of CBX3 deletion on the growth of colon cancer HCT116 was analyzed by EdU proliferation assay. **P<0.01 compared with control. www.impactjournals.com/oncotarget 19936 Oncotarget important for cell cycle G1-S phase transition, including in tumor growth between HCT116-B3 and HCT116-WT CDK2, CDK4, CDK6, p16, p21, CDH3, CDC25A, (Figure 4A, 4B, 4C and 4D). These results suggest that CCND1, CCNE1, E2F1 and E2F3 in HCT116-B3 and CBX3 accelerates tumor formation in vivo. HCT116-WT cells (Figure 3A). We found the mRNA We had identified that the deletion of CBX3 abundances of CDK6 and p21 consistently increased increases CDK6 and p21 expression in colon cancer in the deletion of CBX3 cells (Figure 3B). Immunoblot